Genetic and Physical Mapping of QTLs for Fruit Juice Browning and Fruit Acidity on Linkage Group 16 in Apple  

Takuya Morimoto1 , Koki Yonemushi2 , Hironori Ohnishi2 , Kiyoshi Banno3
1. Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2. Graduate School of Agriculture, Shinshu University, Minami-minowa, Nagano 399-4598, Japan
3. Faculty of Agriculture, Shinshu University, Minami-minowa, Nagano 399-4598, Japan
Author    Correspondence author
Tree Genetics and Molecular Breeding, 2014, Vol. 4, No. 2   doi: 10.5376/tgmb.2014.04.0002
Received: 26 Nov., 2014    Accepted: 29 Dec., 2014    Published: 29 Dec., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Morimoto et al., 2014, Genetic and Physical Mapping of QTLs for Fruit Juice Browning and Fruit Acidity on Linkage Group 16 in Apple, Tree Genetics and Molecular Breeding, Vol.4, No.2 1-10 (doi: 10.5376/tgmb.2014.04.0002)

Abstract

Fruit juice browning and fruit acidity, which are important characteristics for the determination of marketability and processability of apple fruit, are becoming major targets for apple breeding. To identify the genetic basis of these two traits, quantitative trait locus (QTL) analysis was carried out using a 79 progenies of ‘Fuji’ × ‘Maypole’ F1 population. The maternal parent ‘Fuji’ was characterized by low acidity and high browning, whereas the paternal parent ‘Maypole’ showed opposite phenotypes—high acidity and low browning—resulting in an F1 population with a wide range of phenotypes. QTL analysis identified the major QTLs for both traits on the upper part of linkage group (LG) 16 of ‘Fuji’. The effects of these QTLs explained 57.5% and 49.7 % of observed variation in fruit juice browning and fruit acidity, respectively. These two QTLs co-segregated with each other, with the allele for high acidity associated with the allele for low browning and vice versa in this population. To physically map the QTL region, recombinant progeny were genotyped with newly designed DNA markers; both QTLs were delimited to a 514-kb region including 105 annotated genes on apple chromosome 16. Several candidate genes were predicted in this region and their associations with fruit juice browning and fruit acidity were considered

Keywords
DNA marker; Fruit quality; Genetic analysis; Malus×domestica; QTL analysis

Because of its importance to the marketability of apples (Malus×domestica Borkh.), fruit quality improvement is a major goal of apple breeding programs worldwide. Fruit quality is complex and comprises many traits, including fruit size, texture, fruit acidity, soluble solid contents, and fruit juice browning. In addition, the high levels of heterozygosity present in cultivated apple (Velasco et al., 2010) hinder prediction of quantitative phenotypes of progeny derived from controlled crosses. In breeding programs, interest has increasingly focused on the use of molecular markers linked to important fruit characteristics. For these reasons, quantitative trait locus (QTL) analysis to identify QTLs co-segregating with fruit quality traits has become popular as a means to develop marker-assisted breeding programs and to unravel the mechanisms underlying the genetic basis of important characteristics in apple (Chagné et al., 2012; Guitton et al., 2012; Kenis et al., 2008; Liebhard et al., 2003).

With respect to apple fruit quality, attention has focused on two important characteristics: fruit acidity and fruit juice browning. Organic acid levels greatly affect the fruit taste and flavor of a given cultivar as well as its suitability for dessert or processing purposes (Kingston 1992). Fruit acidity also determines whether or not fruit should be consumed immediately and how long fruit can be stored: acidity gradually decreases during storage, and an excessive decrease in acidity reduces the eating quality of the fruit (Iwanami et al., 2012). In mature apple fruit, malic acid is the predominant organic acid, although other organic acids such as citric acid, fumaric acid, and quinic acid are also detectable (Zhang et al., 2010). The major locus controlling apple fruit acidity has been mapped to linkage group (LG) 16 and designated as Ma (malic acid), where Ma corresponds to the dominant high acidity or low pH allele and ma is the low acidity or high pH allele (Maliepaard et al., 1998). The primary role of the Ma locus in determining fruit acidity has also been demonstrated by QTL mapping, as a major QTL has been consistently detected on LG 16 (Kenis et al., 2008; Liebhard et al., 2003; Xu et al., 2011). Recently, Xu et al. (2011) localized the Ma locus to a region of 150 kb on chromosome 16 in the apple genome sequence. In addition, Bai et al. (2012) have identified an aluminum-activated malate transporter (ALMT) gene as a strong candidate for Ma.
Fruit juice browning is closely related to the processability of apple fruit into substances such as juice, sauce, and cut fruit. Because increased browning greatly reduces the marketability of these processed products (Murata et al., 1995), cultivars with a low degree of fruit juice browning are generally desired. Genetic analyses focusing on flesh browning in apple have been performed by several research groups (Kenis et al., 2008; Mellidou et al., 2012; Sun et al., 2014), resulting in the identification of several QTLs. In addition, Gualdo et al. (2013) have reported QTLs for fruit flesh browning and their association with two candidate genes encoding phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO). These studies were all focused on fruit flesh browning rather than fruit juice browning, however, and it is unclear whether or not the two types of browning are regulated by the same genetic mechanism.
In this study, we analyzed a ‘Fuji’ × ‘Maypole’ F1 population to analyze the genetic basis of fruit juice browning and fruit acidity. We identified major QTLs for both traits on LG 16 in ‘Fuji’. In addition, the QTL was physically determined based on genotyping of recombinant progeny, and several candidates located within the QTL region were examined for their associations with fruit juice browning and fruit acidity.
1. Results
1.1 Genetic analysis of fruit juice browning and fruit acidity
For each genotype, average phenotypic values of 3 years from 2011 to 2013 were used in genetic analysis, because fruiting in the ‘Fuji’ × ‘Maypole’ (referred to as Fj×Mp) population was not stable among years and a limited number of individuals produced fruits in each year (e.g., in 2011 fruits of 45 individuals were harvested). Although the number and genotype of fruiting progeny varied among years presumably due to biennial bearing, phenotypic values of fruit juice browning and fruit acidity in the same individuals were almost constant and showed significant correlation (r=0.68~0.70 in fruit juice browning, r=0.80~0.84 in fruit acidity). The degree of fruit juice browning in the Fj×Mp population varied between 1.00 and 5.00, with a mean of 3.48±0.17. The frequency distribution for this trait was bimodal: we observed one peak in the higher range-high and medium browning—representing most of the population, and another in the lower browning range (Figure 1A). The boundary between low and high/medium browning seemed to be around 3.00. On the other hand, fruit acidity ranged from 0.30 to 1.60, with a mean of 0.84±0.03 g/100 mL, and showed a continuous distribution with a single peak around 0.70 g/100 mL (Figure 1B).


Figure 1 Frequency distoributions of fruit juice browning (A) and fruit acidity (B) in F1 population of ‘Fuji’בMaypole’

1.2 QTL mapping
A total of 120 genome-wide SSR markers were used to construct genetic linkage maps for ‘Fuji’ and ‘Maypole’. The ‘Fuji’ map consisted of 103 markers ordered in 17 linkage groups. The map defined 932.3 cM of total map distance and was characterized by an average distance between adjacent markers of 9.1 cM. The ‘Maypole’ map was based on 97 markers on 17 linkage groups. The map defined 894.9 cM of total map distance, with an average distance between adjacent markers of 9.2 cM.
A major QTL [logarism of odds (LOD) = 17.13] for fruit juice browning was detected on the upper part of LG 16 in ‘Fuji’ (Figure 2). This QTL explained 57.5 % of the phenotypic variance in the Fj × Mp population. Two minor QTLs were detected in other linkage groups: one in the middle part of LG 17 in ‘Fuji’ (LOD=3.70; explained phenotypic variance = 9.4 %) and one in the middle part of LG 10 in ‘Maypole’ (LOD=3.49; explained phenotypic variance =10.9 %). A major QTL (LOD=13.50) for fruit acidity, presumably the Ma locus, was detected on the upper part of LG 16 in ‘Fuji’ (Figure 2). This QTL explained 49.7 % of the phenotypic variance. In addition, a minor QTL was detected on the lower part of LG 16 in ‘Maypole’ (LOD=2.81; explained phenotypic variance = 15.6 %). From those results, we attempted to perform QTL analysis using the phenotypic value for each year from 2011 to 2013.


Figure 2 Linkage group 16 of ‘Fuji’ anchoring to physical map of chromosome 16 and LOD profiles of the QTL for fruit juice browning (solid line) and fruit acidity (dashed line)

Although the phenotypic variance fluctuated [36.0% to 62.5% in fruit juice browning, 35.0% to 54.6% in fruit acidity], the significant QTLs tested by the 1000 permutation test at 5% level of significance were also detected on the upper part of LG16 in each year, indicating that those QTLs seems to be stable over 3 successive years.
The two major QTLs for fruit juice browning and fruit acidity were co-localized on LG 16 of ‘Fuji’ and were tightly linked. They were located in the vicinity of the SSR marker NH026a (Figure 2) and flanked by markers CH02a03 and CH05c06. When genotype-phenotype associations for fruit juice browning and fruit acidity were analyzed, the NH026a genotype derived from ‘Fuji’ was clearly associated with both traits in the Fj × Mp population (Figure 3). Progeny possessing the 165-bp genotype of NH026a tended to be distributed on the higher browning and lower acidity side, whereas progeny with the 151-bp genotype generally exhibited lower browning and higher acidity. Furthermore, fruit juice browning and fruit acidity showed a significant negative correlation (r= −0.69), with fruit of higher acidity progeny tending to have lower fruit juice browning, and vice versa (Figure 4).


Figure 3 Genotype-phenotype associations for fruit juice browning and fruit acidity in F1 populations of‘Fuji’בMaypole’


Figure 4 Correlation between degree of fruit juice browning and fruit acidity in F1 populations of ‘Fuji’בMaypole’

1.3 Physical mapping of the QTLs on chromosome 16
Analysis of the SSR markers CH02a03 and CH05c06 identified six recombinants in the Fj × Mp population. Two classes of recombinants were not encountered: those bearing fruit with lower acidity (<0.64 g/100 mL and higher browning (>3.6), as well as those showing higher acidity/lower browning. Screening of contig sequences within the estimated QTL region of 1.4 Mb (CH02a03–CH05c06) identified 18 SSR markers (Table 1). Among the 18 markers, amplification of 15, LG16-769020-1, -769020-2, -837763, -1003247-1, -1159074, -1199876, -1352468, -1437845, -LAR-SSR, -1526288, -1568112, -1661111, -1728791, -1788357 and -1860066, yielded polymorphic fragments that mapped to LG 16 of ‘Fuji’. The 15 sets of SSR markers were then analyzed in the recombinants, and the region of recombination was successfully identified in each recombinant (Figure 5a). From these data, graphical genotypes of the QTLregion were generated. LG16-1199876, NH026a, LG16-1352468, -1437845, -LAR-SSR, -1526288, and -1568112 showed co-segregation with the QTL in the recombinants and the mapping population, suggesting that these markers would be useful for marker-assisted breeding for fruit juice browning and fruit acidity. The QTL regions for both fruit juice browning and fruit acidity were delimited between LG16-1159074 and -1661111 based on key recombinants of Fj × Mp R-6 and Fj × Mp G-38 (Figure 5a). A BLAST search against the apple genome sequence (Genome Database for Rosaceae (GDR) BLAST server (http://www.rosaceae.org) identified two contig sequences containing the two markers—MDC010932.632 for LG16-1159074 and MDC006784.248 for LG16-1661111—that are physically separated by approximately 514 kb (Figure 5b).


Figure 5 Genetic and physical mapping of the QTLs for fruit juice browning and fruit acidity on apple chromosome 16

1.4 Candidate genes for QTLs
To elucidate the genetic mechanisms underlying fruit juice browning and fruit acidity QTLs, the estimated 514 kb region identified in the apple genome was examined by in silico analysis for genes possibly related to fruit juice browning and fruit acidity. A total of 108 genes were predicted in the estimated QTL region, including ALMT (MDP0000252114) and leucoanthocyanidin reductase (LAR) (MDP0000171928).
2 Discussion
In this study, we analyzed the genetic basis of fruit juice browning and fruit acidity using an Fj×Mppopulation. The parents of the studied population had contrasting phenotypes: ‘Fuji’, the leading cultivar in Japanese and global apple production, is characterized by low acidity (0.36 g/100 mL ± 0.02) and a high degree of fruit juice browning (5.00±0.00), whereas ‘Maypole’, which bears small, astringent, sour fruit, has high acidity (1.60 g/100 mL ± 0.04) and a relatively low degree of fruit juice browning (2.28±0.05). In the Fj × Mp population, both fruit juice browning and fruit acidity varied greatly among progeny (Figure 1a; Figure 1b).
The bimodal distribution observed for fruit juice browning suggests the presence of a major QTL controlling this trait in apple (Figure 1a). In contrast, fruit acidity showed a normal distribution with a peak around 0.7 g/100 mL (Figure 1b), a value at the higher end of the acidity range among modern apple cultivars (Iwanami et al., 2012). Mid-parent values of fruit acidity were close to family means of the Fj × Mp population. In crosses in which the acidity of one parent is in a higher category, the distribution of acidity has been reported to shift towards a higher category (Iwanami et al., 2012). The tendency towards high acidity in the Fj × Mp population can thus likely be attributed to the pollen parent ‘Maypole’, which bears fruit of extremely high acidity.
A major QTL for fruit juice browning was detected on LG 16 of ‘Fuji’. In the mapping population used in this study, minor QTLs for fruit juice browning were also detected on LG 10 of ‘Maypole’ and LG 17 of ‘Fuji’. Previous studies have identified several QTLs for flesh browning on LGs 3, 10, 15, 16, and 17 (Kenis et al., 2008; Mellidou et al., 2012; Sun et al., 2014). Although these previous studies were all focused on fruit flesh browning rather than fruit juice browning, the fact that QTLs for the two types of browning were located on the same linkage group suggests that fruit flesh and fruit juice browning are controlled by similar mechanisms. A major QTL for fruit acidity, presumablythe Ma locus, was detected on the upper part of LG 16 in ‘Fuji’ as described in previous studies (Kenis et al., 2008; Liebhard et al., 2003; Xu et al., 2011). Interestingly, this QTL was tightly linked to the fruit juice browning QTL (Figure 2); in addition, a high correlation (r = −0.69**) was found between the two traits (Figure 3 and Figure 4). Those result may provide preliminary data, however, because limited number of genotypes produced fruits in each year (n=45 to 67). The reason why phenotypic variance explained by the QTL on LG16 fluctuated among years is probably due to the variable number of individuals (n=45 to 67) that fruited in each year. A small number of fruited progeny could result in a distorted frequency distribution, and may cause an overestimate of total phenotypic variance in QTL analysis.


Table 1 Newly designed SSR markers on LG16

In the area around the QTL region for both fruit juice browning and fruit acidity, six progeny were identified as recombinants with crossover between SSR markers CH02a03 and CH05c06. Genotyping of recombinants with newly designed SSR markers delimited the QTL to a physical size of 514 kb sandwiched by LG16-1159074 (1.16 Mb) and LG16-1661111 (1.67 Mb) (Figure 5a; Figure 5b). Fine genetic mapping of the Ma locus has recently narrowed down the Ma region to 1.30–1.43 Mb (Xu et al., 2011), consistent with our results. Our study, however, is the first to physically identify the QTL controlling fruit juice browning in apple.
Among the 105 genes predicted within the 514 kb region, ALMT has been identified as a strong Ma candidate because its expression is significantly correlated with fruit titratable acidity (Bai et al., 2012). Furthermore, the cited researchers identified a mutation at 1,455 bp leading to a premature stop codon that truncates the carboxyl terminus of the ALMT protein; the SNP was completely associated with low fruit acidity, suggesting that the natural mutation-led truncation is most likely responsible for the abolished function of Ma for high fruit acidity in apple.
Although candidate genes for fruit juice browning have not been identified in apple, fruit flesh browning QTLs and their association with candidate genes encoding PPO (LG10) and PAL (LG4, 12) have been reported (Guardo et al., 2013). In apple, susceptibility to flesh browning is thought to be the result of complex interplay between the PPO enzyme and polyphenol content (Amiot et al., 1992). The PPO enzyme catalyzes the formation of quinones from polyphenols such as chlorogenic acid and catechin, resulting in browning of fruit flesh and juice (Boss et al., 1995; Falguera et al., 2011). PAL is a key enzyme of the phenylpropanoid pathway that catalyzes the deamination of phenylalanine to trans-cinnamic acid, the latter a precursor to chlorogenic acid, catechin, and anthocyanin (MacDonald and D’Cunha 2007).
Although the two candidate genes reported by Guardo et al. (2013) are not located within the QTL region of chromosome 16, several hypotheses concerning PPO activity and polyphenol content can be advanced based on the candidates within the region. First, PPO activity might be affected by fruit acidity. Malic acid content, the main determinant of fruit acidity in apple, is strongly correlated with fruit juice pH, and QTLs for fruit acidity and pH co-locate on the Ma locus (Bai et al., 2012; Xu et al., 2011; Morimoto et al., unpublished data). Fruit pH appears to be associated with degree of fruit juice browning, as the lower pH of fruit juice tends to be less susceptible to PPO-catalyzed browning (Sun-Waterhouse et al., 2011). We observed a similar trend in the Fj × Mp population, with fruit juice of high-acid fruit progeny tending to exhibit less browning than that from offspring having low acidity (Figure 3 and Figure 4). Thus, the fruit acidity QTL (the Ma locus, ALMT) may be associated with the degree of fruit juice browning through the regulation of pH and PPO activity. This trend, however, does not seems to be true for all apple cultivars, as there are some cultivars that bear low-acidity, low-browning fruit, such as ‘Tsugaru’ (‘Golden Delicious’ × ‘Jonathan’) and ‘Michinoku’ (‘Kitakami’ × ‘Tsugaru’).
Second, LAR located within the QTL region is a possible candidate for determining polyphenol content in apple fruit juice. LAR catalyzes the conversion of leucocyanidins into the flavan-3-ols catechin and epicatechin. Catechin and epicatechin are the building blocks of procyanidins (Bogs et al., 2005) and are substrates for PPO, resulting in fruit flesh/juice browning. MdLAR co-localizes on chromosome 16 with QTLs for phenolic compounds including catechin, epicatechin, and procyanidin (Chagné et al., 2012; Khan et al., 2012a), and LAR expression is significantly correlated with the content of these metabolites (Khan et al., 2012b). LAR is thus another possible candidate gene for the fruit juice browning QTL. Consequently, an understanding of the effects of LAR expression on the degree of fruit juice browning is required for elucidation of the genetic mechanisms controlling fruit juice browning in apple.
3 Materials and Methods
3.1 Plant materials
A cross combinations between apple cultivars ‘Fuji’ and ‘Maypole’ were carried out in 1999, resulting in ‘Fuji’ × ‘Maypole’ F1 population of 79 individuals. Progeny used in this study were grown on their own roots and planted 0.5 m apart in rows separated by 2.0 m at the experimental farm of the Laboratory of Pomology, Faculty of Agriculture, Shinshu University, Nagano, Japan. Orchard management was performed according to commercial practices used in the southern part of Nagano Prefecture.
For DNA extraction, fresh young leaves were collected and stored frozen at −85°C until use. Genomic DNA was extracted from 0.5 g of leaves (fresh weight) using a Plant Genomic DNA Maxi extraction kit (Viogene, New Taipei, Taiwan). The extracted DNA was quantified using a GeneQuant Pro spectrophotometer (Amersham, UK), confirmed by agarose gel electrophoresis, and then diluted to 20 ng/µL with 0.1× TE buffer.
3.2 Phenotypic assessment
Fruit samples were harvested over 3 successive years from 2011 to 2013. Fruit from individual genotypes were picked at maturity, when the ground color at the calyx end had changed from green to yellowish green or creamy, and immediately before commencement of fruit drop in mid-August to late October. The fruits of each individual were harvested several time as they reached maturity.
For phenotypic assessment, five fruits per genotype were randomly selected and assessed for both fruit acidity and fruit juice browning. Crude juice was extracted with a grater from each fruit. After obtaining fruit juice by passing the crude extract through two layers of cheesecloth, 5 mL of this sample was diluted with approximately 30 mL of distilled water. Titratable acidity was determined by titrating the diluted sample with 0.1 N NaOH. Titration results were calculated as malic acid (g) per 100 mL of sample juice. The degree of fruit juice browning was visually evaluated 6 h after juice extraction by two independent investigators and categorized into five classes as follows: 1 (none), 2 (low), 3 (medium), 4 (high), and 5 (extremely high). Phenotypic values of each genotype represented the mean values obtained from the analysis over 3 successive years.
3.3 SSR analysis
A total of 120 SSR markers were selected from previously reported markers (Gianfranceschi et al., 1998; Guilford et al., 1997; Hokanson et al., 1998; Liebhard et al., 2002; Silfverberg-Dilworth et al., 2006; Yamamoto et al., 2002), and each primer was labeled with fluorescent dye (6-FAM, HEX, or NED). PCR amplifications were performed under the following conditions: initial denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 1 min, 60°C~55°C for 1 min, and 72 °C for 2 min, and a final extension at 72 °C for 8 min. Allele sizes of the parental amplicons were determined using an ABI PRISM 310 Automated Fluorescent DNA sequencer (Applied Biosystems, Foster City, CA, USA), and the amplified products from the population were separated on a 6% denaturing polyacrylamide gel containing 8 M urea in 0.5× TBE buffer. Fragments were visualized by silver staining using a Silver Sequence DNA Sequencing system (Promega, WI, USA).
3.4 Genetic linkage mapping and QTL analysis
Segregation analysis based on the pseudo-test cross mapping strategy (Grattapaglia and Sederoff 1994) was performed on 79 progeny of Fj × Mp. Segregation of each marker was scored by the presence or absence of a specific parental band. Genetic linkage maps were constructed using MAPL 98 mapping software (Ukai. 1998). Genetic map distances in centimorgans (cM) were calculated using the Kosambi mapping function (Kosambi. 1944).
QTL analyses were carried out using all markers mapped onto genetic linkage maps of ‘Fuji’ and ‘Maypole’. Windows QTL cartographer version 2.5 (Wang et al., 2010) was used to perform composite interval mapping with a step size of 1.0 cM. The logarism of odds (LOD) threshold for declaring the presence of a QTL was defined by 1,000 permutation tests at a 5% level of significance. The position at which the LOD score curve reached its maximum was used as the estimate of the QTL location. The percentage of the phenotypic variance explained by a QTL was estimated as the coefficient of determination (R2).
3.5 Marker development for physical mapping of QTLs
Sequences of two SSR markers, CH02a03 and CH05c06, which flanked the QTLs on linkage group 16 of ‘Fuji’, were obtained from the High-Quality Disease Resistant Apples for a Sustainable Agriculture website (http://www.hidras.unimi.it/). The markers were BLAST-searched against the genomic sequence of the cultivar ‘Golden Delicious’ using the GDR BLAST server (http://www.rosaceae.org). DNA sequences of the QTL regiondelimited by the two flanking markers were screened for the presence of SSR motifs using Tandem Repeats Finder (Benson et al., 1999). Primer pairs flanking SSR motifs were designed with Primer 3 Plus (Untergasser et al., 2007). PCR amplification and SSR analysis were conducted as described above.
3.6 Candidate gene searching
We searched for predicted gene transcripts within the estimated QTLregion on the ‘Golden Delicious’ genomic sequence using GDR GBrowse. Their locations and putative functions were based on Malus × domestica whole genome v1.0 assembly & annotation, GDR GBrowse.
Author contributions
TM and KB designed the experiment. TMand KY performed experiments. TM, KY and HO analyzed data. TM and KB drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgments
The research was financially supported by the Sasagawa Scintific Reserch Grant from The Japan Science Society.
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